1. Stroma-Free Hemoglobin
Hemoglobin solutions have long been proposed as cell-free oxygen carriers to be used as red cell substitutes in infusional fluids. The interest in these compounds stems from their potential clinical applications. They can be sterilized, easily stored, and used in transfusions without blood-group typing.
Intravenously injected (infused) crude hemolysates and extensive hemolytic processes produced in vivo by immunological reactions involving intravascular lysis of red blood cells, are known to produce a clinical syndrome characterized by disseminate intravascular coagulation. This syndrome is often fatal and is produced by the residual red blood cell walls (stroma) and their fragments, so infused into circulating blood. Stroma-free hemolysates do not show this toxicity (See Rabiner et al, J. Exp. Med., 126:1127 (1967)). As a result, it has been desired to use stroma-free hemoglobin as an oxygen carrier in cell-free transfusional fluids.
However, the use of stroma-free hemoglobin has the following two disadvantages: In vivo, the retention time of the stroma-free human hemoglobin is very short, i.e., it has a half-life on the order of 1-4 hr (see Rabiner et al, supra; and De Venuto et al, Transfusion, 17:555 (1977)). "Half-life" is defined as the time necessary to eliminate 50% of the infused hemoglobin from circulating blood. Further, outside of the red blood cells, hemoglobin has a high affinity for oxygen which, in vivo, would prevent the release, i.e., the transport, of oxygen from hemoglobin to the tissues. These disadvantages are directly the result of the molecular structure of hemoglobin.
Hemoglobin is a tetrameric molecule having a molecular weight of 64,500 Daltons. The tetrameric molecule is formed of two pairs of .alpha. and .beta. subunits. The subunits are held together as a result of ionic, hydrophobic and Van der Waals forces, and not as a result of covalent bonds. When hemoglobin is oxygenated, i.e., combined with oxygen, it readily forms .alpha.-.beta. dimers having a molecular weight of 32,250 Daltons. These dimers are not retained in vivo by the kidneys and are eliminated through the urine. In the rat, untreated hemoglobins are rapidly eliminated in the urine and have a half retention time of only 40-50 min. Urine elimination is the result of the dissociation of tetrameric hemoglobins into dimers.
The tetrameric structure of hemoglobin also provides a binding site for 2,3-diphosphoglycerate. Inside red blood cells, 2,3-diphosphoglycerate combines with hemoglobin in order to decrease its oxygen affinity to a level compatible with oxygen transport. The binding of 2,3-diphosphoglycerate and hemoglobin is purely electrostatic and no stable covalent bonds are formed. Thus, when red blood cells are ruptured and 2,3-diphosphoglycerate is not retained inside the cells by the cell wall, it is released from hemoglobin. As a result, hemoglobin acquires a higher oxygen affinity. This prevents the transport of oxygen from hemoglobin to the tissues. The level of this higher affinity is sufficient such that the oxygen affinity can be considered "non-physiological".
Because of the many appealing qualities of hemoglobin, i.e., its ability to reversibly bind oxygen, the low viscosity of a hemoglobin solution and its easy preparation and storage for long periods of time, various attempts have been made in order to overcome the above described disadvantageous characteristics of stroma-free hemoglobin. These various attempts are discussed in more detail below.
2. Chemical Treatments for Preventing the Formation of Dimers
The formation of .alpha.-.beta. dimers, which are not retained in vivo, can be prevented by coupling the tetrameric molecules of hemoglobin with large molecular weight matrices, ranging from 20,000 to 275,000 Daltons. For example, matrices such as dextran (see Tam et al, Can. J. Biochem., 56:981 (1978); and Bonneaux et al, Experientia, 37:884 (1981)) and hydroxyethyl starch (see Baldwin et al, Tetrahedron, 3:1723 (1981); and U.S. Pat. Nos. 4,412,989, 4,900,816, 4,650,786 and 4,710,488) have been employed. This coupling prevents the elimination of hemoglobin in vivo from the kidneys by way of the urine. Other types of polymeric coupling employing collagen, collagen degradation products, and gelatin as a supporting matrix have also been employed (see U.S. Pat. No. 2,591,133; U.S. Pat. No. 3,057,782; and Bowes et al, Biochem. Biophys. Acta., 168:341 (1968)). However, the oxygen affinity of the resulting coupled hemoglobin is even higher than that of stroma-free hemoglobin, and thus hemoglobin coupled in this manner cannot be advantageously employed as an oxygen transport medium.
Other known treatments for preventing the formation of .alpha.-.beta. dimers are based on reactions which polymerize the tetrameric molecules of hemoglobin to form so-called "polyhemoglobins". Polyhemoglobins can be obtained using bifunctional reagents, such as glutaraldehyde (see Hopwood et al, Histochem. J., 2:137 (1970)) or diimidate esters (see Mock et al, Fed. Proc., 34:1458 (1975); and U.S. Pat. No. 3,925,344). These bifunctional reagents form covalent bonds between the amino groups present on the surface of different hemoglobin molecules producing intermolecular cross-links. There are 40 or more of such amino groups belonging to lysyl residues on the surface of mammalian hemoglobins (44 in human hemoglobin). Thus, a large number of possible combinations of hemoglobin molecules occur. As a result, the polyhemoglobin reaction products are a heterogeneous mixture of various molecular species which differ in size and shape. The molecular weights thereof range from 64,500 to 600,000 Daltons. Longer retention times in vivo are obtained using polyhemoglobins.
Besides the various treatments discussed above which result in formation of heterogeneous mixtures of polyhemoglobin, reagents have been developed which are capable of producing an internal cross-link of the hemoglobin subunits with little or no formation of polyhemoglobins. More specifically, the formation of cross-links between the .beta. subunits of hemoglobin using 2-N-2-formylpyridoxal-5'-phosphate and borohydride and bis-pyridoxal polyphosphates have been carried out (see Benesch et al, Biochem. Biophys. Res. Comm., 62:1123 (1975); and Benesch et al, Biochem. Biophys. Res. Comm., 156:9 (1988)). The oxygen affinity of the. thus treated hemoglobin is decreased to levels similar to that of normal blood. However, the reagents employed therein are very difficult and costly to synthesize, and thus these methods are disadvantageous.
Other reagents have been employed in order to effect internal cross-linking of the hemoglobin subunits. These reagents are commonly known as "diaspirins". Diaspirins are diesters of 3,5-dibromosalicyl containing succinyl, fumaryl or other dicarboxylic acid residues. These reagents produce covalent cross-links between two .beta. or two .alpha. subunits of an individual hemoglobin molecule. While better results are obtained using liganded (oxy- or carboxy-) hemoglobin, such a treatment does not sufficiently affect the oxygen affinity characteristics of stroma-free hemoglobin, and thus can not be advantageously employed (see Walder et al, J. Mol. Biol., 141:195 (1980); and U.S. Pat. Nos. 4,061,736, 4,001,200, 4,001,401, and 4,053,590).
It should be stressed that according to pertinent literature, i.e., Walder, Biochem., 18:4265-4270 (1779); Walder et al, J. Mol. Biol., 141:195 (1980); and Zaugg, J. Biol. Chem., 255:2816-2821 (1980), activated dicarboxylic acid of increasing length, above that of the 4-carbon chain of succinic and fumaric acid, show a progressively lower reactivity with both liganded and unliganded hemoglobin so that activated sebacic acid (10-carbons long) produces no reactions with human hemoglobin.
In U.S. Pat. Nos. 4,473,496 and 4,857,636, linear .alpha.-.omega. or heterocyclic polyaldehydes containing negatively charged groups are described as suitable for both decreasing the oxygen affinity of hemoglobin and for producing inter- and intramolecular cross-linking of hemoglobin. These reagents include carbohydrate-containing molecules, such as raffinose, and mono- and polyphosphorylated nucleotides partially oxidized with periodate, so as to obtain aldehydic groups. The coupling reaction is based on the formation of Shiff bases of the aldehydic groups with the amino groups of the hemoglobin molecule. The Shiff bases are then transformed into covalent bonds by reduction with sodium or potassium borohydride, or another strong reducing agent.
In U.S. Pat. No. 4,584,130, cross-linking of hemoglobin with bifunctional reagents is disclosed. The reagents disclosed therein are based on an electron withdrawing group which modulates the reactivity of two peripheral active groups. However, the electron withdrawing group remains within the cross-linking bridge after the reaction. In the reagent of the present application, when an electron withdrawing group is employed, it is present in the leaving group only (e.g., 3,5-dibromosalicyl), and therefore it does not remain in the cross-linking bridge after the chemical reaction of the activated carboxyls with the amino groups of the protein.
3. Chemical Treatments for Decreasing the Oxygen Affinity of Stroma-Free Hemoglobin
The most widely used chemical modification of stroma-free hemoglobin so as to decrease the oxygen affinity thereof employs the use of pyridoxal-5'-phosphate and sodium or potassium borohydride (see Bensch et al, Biochem., 11:3576 (1972)). The resulting product is commonly referred to as "PLP-hemoglobin" and has satisfactory oxygen affinity, i.e., oxygen affinity very near that of the red cells present in normal blood.
Other known chemical modifications of hemoglobin have been carried out using phosphoric acid derivatives of carbohydrates (e.g., glucose-6-phosphate) (see McDonald et al, J. Biol. Chem., 254:702 (1979)); carbamylation (see Manning, Meth. Enz., 76:159 (1981)) and carboxymethylation (see DiDonato et al, J. Biol. Chem., 258:11890 (1983)). In each of these treatments, the amino-terminal end of the .beta. subunit of hemoglobin is permanently substituted with the above described reagents.
In addition, none of these chemical treatments discussed in this section stabilize the tetrameric structure of hemoglobin so as to prevent the formation of .alpha.-.beta. dimers. Thus, the resulting hemoglobins do not have prolonged retention times in vivo.
4. Combined Chemical Treatments for Preventing the Formation of .alpha.-.beta. Dimers and Decreasing the Oxygen Affinity of Stroma-Free Hemoglobin
As discussed above, the production of physiologically competent stroma-free hemoglobin-based oxygen carriers necessitates two separate treatments. That is, one treatment is necessary for preventing the formation of .alpha.-.beta. dimers in vivo and a second treatment is required for decreasing its oxygen affinity. The most widely employed combination of treatments is that of reacting glutaraldehyde with PLP-hemoglobin to form pyridoxylated polyhemoglobins (see Seghal et al, J. Surg. Res., 30:14 (1981)). Intramolecular cross-linking of PLP-hemoglobin has also been obtained using diaspirins (see Tye et al, Prog. Clin. Biol. Res., 22:41 (1983)).
It should be noted that only stroma-free hemolysates or washed red blood cells are utilized in the above-cited articles. That is, purification procedures for isolating the hemoglobin component of the stroma-free hemolysates are not described therein. Thus, what is defined as stroma-free hemoglobins therein is in actuality stroma-free hemolysates.
More specifically, about 95% of the hemolysate components is hemoglobin. The remainder consists of proteins and polypeptides whose pharmacological and immunological toxicity is not known. When used for infusion in animals, several grams of hemolysate-containing hemoglobin are injected. Thus, undesirably, hundreds of milligrams of substances of unknown biological activity are also infused into animals when employing a hemolysate.
It should also be noted that in the above-cited references, purification procedures for isolating the desired hemoglobin products from the reaction mixture are not described therein. It is impossible to avoid the presence of overreacted and underacted hemoglobins in the reaction mixtures. These products do not have the desired functional and molecular characteristics.
For the above reasons, it is advantageous to perform chemical treatments on purified hemoglobins, and then to purify the product of the reaction.
Recently, reagents which present a clear advantage over previously employed long chain divalent reagents for producing intramolecular cross-linked hemoglobin have been described (U.S. Pat. No. 5,387,672). These cross-linking reagents give rise to cross-linked hemoglobin which has a lower oxygen affinity, and can be obtained in a much higher yield. While the cross-link is still intramolecular, the hemoglobin thereof has been found to be stable, not only against dissociation, but also against physical agents like heat, pH and aging. Thus, the formation of ferric hemoglobin is greatly retarded with the cross-linked hemoglobin. This allows the use of high temperature heat treatments for eliminating pathogens, the use of lyophilization procedures and to effect storage in liquid form in the cold and at room temperature.
Prevention of dimer dissociation in intramolecularly cross-linked tetramers, prevents glomerular filtration and prolongs the intravascular retention time of the protein (Urbaitis et al, J. Lab. Clin. Med., 117:115 (1991); Bucci et al, Biomat. Art. Cells & Immob. Biotech., 20(2-4):243 (1992); Matheson-Urbaitis et al, J. Lab. Clin. Med., 126:250 (1995); and Ulatowski et al, Am. J. Phys., 270:H466-H475 (1996)). Because nephrotoxicity of filterable (dissociable) hemoglobin results, in part, from intratubular precipitation and hemin formation (Fitzpatrick et al, Clin. Res. 42:220 (1994); and Paller, Am. J. Physiol., 255:F539 (1988)), these non-filtered tetramers are expected to be less or non-nephrotoxic.
These stable tetramers of hemoglobins are not filtered at the glomerulus, however this does not rule out their entry into the renal interstitium where other adverse reactions might occur or result after cellular uptake (Urbaitis et al, J. Lab. Clin. Med., 117:115 (1991)). Since these stable hemoglobins have a plasma half time in the rat of about 4 hrs, less than the 6 hrs for albumin, they must have a significant degree of migration into tissues. Post glomerular capillaries are of the fenestrated type with pores having a diameter of 400 to 600 .ANG., and have a high degree of permeability to small solutes and water. Similar to other capillaries, they function as if they have large and small pores (Rippe et al, Physiol. Rev., 74:163 (1994)). Experimental data indicate that they have a high reflection coefficient for albumin. However, albumin does pass into renal lymph ostensibly through the "large pores". The passage of stable tetrameric hemoglobin into posterior lymph was has been reported (Bleeker et al, J. Lab. Clin. Med., 113:151 (1989)), suggesting that these materials pass over large pores in other capillaries in the body. It has been found that these stable tetramers also appear in renal lymph, even in the complete absence of hemoglobinuria.
The passage of a molecule through the "large pores" is affected by the size of the molecule (Larson et al, Am. J. Physiol., 253:F180 (1987)). Sieving curves generated by relating interstitial concentration to renal venous concentration of inulin, myoglobin, horse radish peroxidase and albumin indicate a retarding effect of molecular size (Aukland et al, Am. J. Physiol., 266:F175 (1994)). The large pore reflection coefficient of albumin with MW 64,000 Da was found to be 0.24 (maximum reflection is 1.0) while that for .gamma.-globulins with MW 156,000-0160,000 Da was 0.44 (Larson et al, Am. J. Physiol., 253:F180 (1987)).
Cross-linked hemoglobin with a similar molecular weight and diameter to albumin would also be expected to pass across "large pores" and enter into the renal interstitium. Based on available data, an increased reflection coefficient would be expected to result as the size of the hemoglobin molecule is increased by polymerization. For example, it would be expected that a polymer of 4 tetrameric Hb units would have a molecular size near 250,000 Da, and would therefore be further retarded in passage across the renal capillaries. Thus, the production of polymerized hemoglobins with a larger molecular diameter (radius) appears to be a rational goal.
Two oxygen carriers, i.e., intramolecularly cross-linked tetramers based on human and bovine hemoglobins, have been extensively characterized and used in physiological trials (Urbaitis et al, J. Lab. Clin. Med., 117:115 (1991); Bucci et al, Biomat. Art. Cells & Immob. Biotech., 20(2-4):243 (1992); Matheson-Urbaitis et al, J. Lab. Clin. Med., 126:250 (1995); and Ulatowski et al, supra). They are stabilized by a covalent cross-link between the .beta.82 lysines of the opposite subunits in the .beta.-cleft of hemoglobin. The linker is either a fumaryl residue (4 carbon long) in bovine hemoglobin or a sebacoyl residue (10 carbon long) in human hemoglobin. The intramolecular cross-link prevents the dissociation of the molecule into dimers and brings the oxygen affinity to a physiologically compatible level. As the molecules do not dissociate into dimers, they are not eliminated in the urine and their intravascular retention time is prolonged to about 4 hrs in the rat and 6 hrs in the cat (Urbaitis et al, J. Lab. Clin. Med., 117:115 (1991); and Ulatowski et al, supra).
These stable tetramers are relatively small and cannot be used at concentrations higher than 6-7%, because of their colloid oncotic pressure (COP) activity per gram. Thus, these infusional fluids have half the oxygen capacity of blood. Therefore, it is very important to develop polymeric hemoglobins which would produce longer retention times, would not migrate into the interstitial fluid and would carry more oxygen per unit of oncotic pressure. The low COP/O.sub.2 capacity ratio would allow the production of infusional fluids with very high oxygen capacity, even higher than in normal blood, yet maintaining COP in the usual physiological range.
Polymerized hemoglobins are commonly obtained by treatment with either glutaraldehyde (Sehgal et al, Transfusion, 23:158 (1983)), or raffinose (More et al, Biomat. Art. Cell & Immob. Biotech., 20:293 (1992)). With both procedures, the chemical reaction is random and difficult to control. The obtained polymers are highly heterogeneous, with molecular size distributed over a wide range. The starting material is usually natural, dissociable hemoglobin. This adds to the heterogeneity, because polymerization involves both the tetrameric and dimeric forms of hemoglobin. Both kinds of polymers have been used in initial clinical trials without any major side effects (Current Issues in Blood Substitutes Research and Development, San Diego, Calif., March (1995)).
Glutaraldehyde is a bifunctional reagent which produces polymers by forming Schiff bases with the amino groups of lysyl residues in adjacent molecules. The Schiff base is not a stable covalent bond. In its reduced form it is more stable. However, it is not clear whether reducing processes can be used which will reduce 100% of the Schiff bonds. If only a few % of the Schiff bases are not reduced, during metabolic processes in vivo, highly toxic glutaraldehyde can be released.
Polymerization with periodate oxidation and raffinose must be performed in an atmosphere of carbon monoxide and the degraded raffinose linker may still produce a metabolic "indigestion" in vivo.